1. Field of the Invention
The invention relates to particulate systems loaded with cyclosporin (also spelled xe2x80x9ccyclosporinexe2x80x9d) or cyclosporin derivatives of natural and/or synthetic origin, which said systems have improved biopharmaceutical properties for cyclosporins in vivo, improved quality (fineness and homogeneity of the particles, drug inclusion) and improved physical stability of the particulate formulation (no aggregation or gel formation).
2. Description of the Related Art
Cyclosporins are cyclic oligopeptides. They are a group of natural oligopeptides ranging from cyclosporin A to cyclosporin Z. Synthetic derivatives have also been described (SDZ IMM 125, the hydroxyethyl derivative of D-serine-8-cyclosporin).
Cyclosporin A is a lipophilic molecule consisting of 11 aminoacids. It is obtained by fermenting mushrooms. Its molecular weight is 1203.
Commercial products: Sandimmun(copyright), Sandimmun Optoral(copyright) (outside Germany=Sandimmun Neoral(copyright)) [A. Meinzer, E. Mxc3xcller, J. Vonderscher, Perorale Mikroemulsionsformulierungxe2x80x94Sandimmun Optoral(copyright)/Neorale(copyright), in: Pharmazeutische Technologie: Moderne Arzneiformen, R. H. Mxc3xcller and G. E. Hildebrand (eds.), Wissenschaftliche Verlagsgesellschaft Stuttgart, 169-177, 1998]. Cyclosporin A is preferably used as an immunosuppressant after organ transplants. Other fields of use are autoimmune diseases, psoriasis and diabetes. All the cyclosporins (both natural and synthetic) can be used in the present invention.
The cyclosporins are highly lipophilic substances and poorly soluble in water (e.g. cyclosporin A:  less than 0.004% m/V in water at 25xc2x0 C.). Their high lipophilia and very poor solubility in water constitute the main problems in producing a suitable pharmaceutical preparation. In view of their better solubility in fatty oils and alcohol, Sandimmun(copyright) was developed with these ingredient as solubility enhancers for oral use in the form of an emulsion concentrate. The said emulsion concentrate consists of 100 mg cyclosporin dissolved in 1 ml of a mixture of oil, ethanol and an emulsifier, namely macrogol glycerol trioleate linolate. The concentrate must be diluted before use, for example by stirring it with a spoon into cold milk, cocoa or fruit juice. This non-standardised, inefficient mixing procedure results in the formation of a coarse non-homogenous oil/water emulsion with a relatively large droplet size. Its bioavailability after oral administration varies in vivo, in extreme cases between 10 and 60% [T. Beveridge, A. Gratwohl, F. Michot et al., Curr. Ther. Res., 30 (5), 1981].
In addition to the oral solution formulation, Sandimmun(copyright) is also available in capsule form. The capsules contain 25 mg/100 mg cyclosporin A dissolved in a mixture of oil, ethanol and emulsifier. In this case, the oily preparation is dispersed in the stomach by peristaltic movements. Here again, this is an inefficient oil dispersion procedure.
Alternatively, the oily phase loaded with cyclosporin A has been treated in further experiments with high-pressure homogenisation. This process produced a finer O/W emulsion [Dietl, H., Pharmaceutical preparation containing cyclosporin(s) for oral administration and process for producing same, U.S. Pat. No. 5,637,317, 1997]. However, this patent contains no data relating to the physical stability of the homogenised emulsion during storage, nor in vivo data demonstrating that the homogenisation process can lead to increased bioavailability. It is known that when cyclosporin is dispersed in the oily phase of an O/W emulsion it precipitates after a few days, forming large crystals in the emulsion by crystallisation of the drug, or the cyclosporin that has exited from the oily phase floats, forming an edge or film on the surface. This problem is known, for example, in the case of the Sandimmun(copyright) oral emulsion. It is also known that the incorporation of a drug into the oily phase of an O/W emulsion can reduce the physical stability of the said emulsion in view of its tendency to coalesce [S. S. Davis, Pharmaceutical aspects of i.v. fat emulsions, J. Hosp. Pharm., 32, 149-170, 1974]. The small size of the droplets is not the only critical factor that causes increased bioavailability of cyclosporin. Homogenisation of the emulsion alone does not automatically increase bioavailability, as normal cyclosporin A re-absorption is also largely influenced by the secretion of bile salts [A. Meinzer, E. Mxc3xcller, J. Vonderscher, Perorale Mikroemulsionsformulierungxe2x80x94Sandimmun Optoral(trademark)/Neoral(trademark), in: Pharmazeutische Technologie: Moderne Arzneiformen, R. H. Mxc3xcller and G. E. Hildebrand (eds.), Wissenschaftliche Verlagsgesellschaft Stuttgart, 1998]. Apart from the extent of bile salt release, food intake also constitutes a significant factor during drug absorption which can influence the bioavailability of cyclosporin. The release of drugs from emulsions also depends on the coefficient of distribution. This influence is difficult to control in order to obtain protracted, non-variable blood levels. These disadvantages of O/W emulsions (ie. coarse emulsions) have already been described for other cyclosporin emulsions [e.g. A. Tibell et al., Cyclosporin A in fat emulsion carrier. Immunosuppressive effect in vitro, J. Immunolo. 35, 231-236, 1992].
The next stage of development was the replacement of the Sandimmun(copyright) coarse emulsion formulation with the Sandimmun Optoral(copyright) microemulsion. The result was that absorption of cyclosporin A became nearly independent of bile salt secretion [A. Meinzer, E. Mxc3xcller, J. Vonderscher, Perorale Mikroemulsionsformulierungxe2x80x94Sandimmun Optoral(trademark)/Neoral(trademark), in: Pharmazeutische Tecnologie: Moderne Arzneiformen, R. H. Mxc3xcller and G. E. Hildebrand (eds.), Wissenschaftliche Verlagsgesellschaft Stuttgart, 176, 1998]. A microemulsion does not contain separate droplets, but is a xe2x80x9ccritical solutionxe2x80x9d [B. W. Mxc3xcller, Mikroemulsionen als neue Wirkstofftragersysteme, in Pharmazeutische Technologie: Moderne Arzneiformen, R. H. Mxc3xcller and G. E. Hildebrand (eds.), Wissenschaftliche Verlagsgesellschaft Stuttgart, 161-168, 1998; B. W. Mxc3xcller, H. J. Franzky, C. J. Kxc3x6lln, U.S. Pat. No. 4,719,239, 1988]. Oral administration of the cyclosporin A microemulsion reduces the variability of absorption, although it produces high initial blood level peaks, well above the limit of 1000 ng/ml (FIG. 1, right). The said blood level peaks must be eliminated in an optimised preparation.
There are no effective topical preparations with cyclosporin A designed for topical treatment (e.g. psoriasis). In the literature, cyclosporin is said to have a topical action in theory (Clinical Report, Servizio de Medicina, Hospital del Cobre, Rancagua. Chile, Rev. Med. Chil. 1994, vol. 122; 1404-7]. However, its efficacy was only observed after six months"" treatment; moreover, dimethyl sulphoxide had to be used as solvent at the concentration of 50%, which is unacceptable for a treatment like that of psoriasis. Theoretical therapeutic efficacy was also reported in another protocol (Intralesional cyclosporine for psoriasis. Relationship of dose, tissue levels and efficacy. J. Gajardo, J. Villaseca, Arch. Dermatol. 1992, vol. 128; 786-790). Topical application had no effect in this case, which demonstrates the importance of a suitable pharmaceutical form. Intralesional injections were therefore performed, and demonstrated the theoretical efficacy of the substance, although its use proved totally impracticable for treatment purposes.
The main technical and biopharmaceutical problems of cyclosporin A formulations at present are:
1. the pharmaceutical quality and physical stability of the preparation (e.g. coarse emulsion, formation of cyclosporin A crystals and coalescence),
2. high variability of blood levels,
3. blood level peaks substantially  greater than 1500 or  greater than 1200 or  greater than 1000 ng/ml, which cause toxic side effects of various kinds,
4. ineffective topical formulations.
The objective of this invention is to eliminate the above said problems regarding the preparation and action of cyclosporin formulations. Alternatively, the invention can be used to prepare formulations that allow cyclosporin transport in the dermis in order to produce an effective topical treatment.
This objective is achieved by a drug carrier comprising solid lipid particles loaded with cyclosporin, and its use.
Absence of blood level peaks and extended release time are obtained with the use of fine particles of solid lipids. Unlike the liquid oily phase of an OW emulsion, because of the solid lipid matrix the release profile can be controlled by diffusion of the drug in the disintegrating lipid matrix.
In view of the lipophilic nature of the cyclosporin drug, lipids are preferred as the ideal candidates for incorporation into lipid particles as matrix material. Unlike polymer particles, lipid particles can be prepared on an industrial scale by high-pressure homogenisation [R. H. Mxc3xcller and S. J. Lucks, Europ. Patent EP 0 605 497 B1, 1996].
The drug carrier is preferably prepared without halogenated organic solvents, and in particular without organic solvents at all.
The two basic preparation techniques are hot homogenisation and cold homogenisation [C. Schwarz, W. Mehnert, J. S. Lucks, R. H. Mxc3xcller, Solid lipid nanoparticles (SLN) for controlled drug delivery. I. Production, characterization and sterilization, J. Controlled. Rel., 30, 1994, 83-96].
In the hot homogenisation technique, the drug is first dissolved or finely dispersed in the molten lipid. The fat loaded with the drug is then dispersed in a hot solution of emulsifier at temperatures higher than the melting point of the lipid, and stirred to obtain a pre-emulsion. This coarse pre-emulsion is then dispersed by high-pressure homogenisation at pressures between 100 and 1500 bars in one or more homogenising cycles. High-pressure homogenisation also takes place at temperatures higher than the melting point of the lipid matrix. The nanoemulsion thus obtained is cooled, and the fat recrystallises to form solid lipid nanoparticles (SLN).
When the cold homogenisation technique is used, the fat remains in the solid state, ie. homogenisation takes place at temperatures lower than the melting point of the lipid. The fat containing the drug is reduced to microparticles in advance with a grinder such as a mortar grinder. The lipid particles thus obtained are then dispersed in a cold solution of emulsifier and homogenised by high-pressure homogenisation. The shearing forces and cavitation are strong enough to reduce the size of the solid lipid and form solid, ultrafine lipid particles called xe2x80x9csolid lipid nanoparticlesxe2x80x9d.
Both techniques have been used in the present invention to prepare lipid formulations loaded with cyclosporin.
In particular, the drug carrier in accordance with the invention comprises particles, with or without surfactants, of a lipid or mixture of lipids having a particle size of between 10 nm and 100 xcexcm which are solid at ambient temperature, in which the particles in the main population have a mean particle diameter of between 40 nm and 100 xcexcm and can be obtained by dispersing an internal phase (lipid phase) in a dispersion medium (water, aqueous solution or a liquid miscible with water) in molten or softened form, or by dispersing an internal phase (lipid phase) in a dispersion medium in solid form and reducing the solid phase in size into fine particles before the dispersion process.
The particles in the solid state at ambient temperature preferably have a diameter of between 10 nm and 10 xcexcn when prepared by high-pressure homogenisation; in this case the particles in the main population have a mean PCS particle diameter of between 40 nm and 1000 nm. The particles in the main population preferably have a mean particle diameter of between 100 nm and 500 nm, and the PCS particle diameters can be adjusted to between 40 nm and 100 nm by means of suitably selected process parameters and additives. The main population therefore constitutes the majority of the particles in the population.
Other size reduction procedures suitable for preparation of the drug carrier in accordance with the invention are high-speed stirring, exposure to ultrasound or a grinding process, especially with the use of jet-stream or airstream mills in which the solid particles in the main population have a mean particle diameter of between 0.5 xcexcm and 100 xcexcm (detected by laser diffractometry).
In order to keep the volume of the final preparation designed for oral administration sufficiently small, it is preferable to load the lipid matrix with 20% of cyclosporin A. Earlier experiments demonstrated that if the lipid matrix is strongly loaded with drug, for example with a 20% load of tetracaine, very coarse dispersions are obtained. Particle aggregation was promoted and a gelling process took place in the first few hours after preparation [A. zur Mxc3xchlen, C. Schwarz, W. Mehnert, Solid lipid nanoparticles (SLN) for controlled drug deliveryxe2x80x94drug release and release mechanism, Eur. J. Pharm. Biopharm., in press, 1998]. It was therefore expected that the manufacture of a dispersion of lipid particles with cyclosporin with relatively high drug loading of the lipid matrix of nanometric size, with high dispersity (=smal particle size) and sufficient physical stability, would have been very unlikely. However, the opposite effect was observed. The particle size of the cyclosporin-loaded lipid dispersion reduced as the cyclosporin content in the lipid matrix increased, and at the same time the polydispersity of the dispersion declined. Cyclosporin aids the formation of ultrafine lipid particles of small size with a high level of homogeneity. The addition of cyclosporin to the lipid matrix increases the pharmaceutical quality of the lipid nanoparticle dispersion; the optimum stability value is produced with a 20% (V/V) drug load.
The drug carrier in accordance with the invention therefore has an internal phase (lipid phase) content amounting to between 0.1% and 40% (m/m), and in particular between 1% and 20% (m/m), of the complete formulation.
Drug-loaded lipid nanoparticles have so far been generally described as physically unstable [C. Schwarz, Feste Lipidnanopartikel: Herstellung, Charakterisierung Arzneistoffinkorporation und-freisetzung, Sterilisation und Lyophilisation, Dissertationsschrift, Freie Universitxc3xa4t Berlin, 1995]. The destabilisation of the said formulation takes place in three stages:
1. The lipid particles aggregate, as can easily be deduced from the increase in mean diameters.
2. The viscosity of the dispersion increases considerably, indicating progressive contact between the aggregates.
3. The lipid nanoparticles initially have a slightly creamy consistency; later, they form solid gels.
The gel consists of a lattice of lipid particles. It has been observed that gel formation is accompanied by a reduction in the fraction of variant xcex1 and a simultaneous increase in the fraction of variants xcex2 and xcex2xe2x80x2 [C. Freitas, R. H. Mxc3xcller, Long-term stability of solid lipid nanoparticles (SLN(trademark)). II. Influence of crystallinity of the lipid and shear forces, submitted to Eur. J. Pharm. Biopharm. 1997].
Physically stable lipid particle dispersions (absence of aggregate and gel formation) presented little reduction, or even a slight increase, of the fraction of variant xcex1 during storage. In this case, no lipid portion was converted into variant xcex2 or xcex2xe2x80x2 [C. Freitas, R. H. Mxc3xcller, Long-term stability of solid lipid nanoparticles (SLN(trademark)). II. Influence of crystallinity of the lipid and shear forces, submitted to Eur. J. Pharm. Biopharm. 1997]. It is known that the presence of pharmaceutical substances in the lipid matrix aids crystallisation in the most stable variant xcex2 or xcex2xe2x80x2 [B. Siekmann, Untersuchungen zur Herstellung und zum Rekristallisationsverhalten schmelzemulgierter i.v. applizierbarer Glyceridnanopartikel, Dissertationsschrift, Techn. Universitxc3xa4t Carolo-Wilhelmins zu Braunschweig, 1994].
In the case of pure lipid particles, which normally remained in the liquid state after production and presented a slower recrystallisation process after days or weeks, recrystallisation could be accelerated by adding pharmaceutical substances. It was therefore expected that the incorporation of cyclosporin into the lipid matrix would destabilise the lipid particle dispersion. Surprisingly, however, the opposite occurred. The formation of variant xcex2 or xcex2xe2x80x2 was inhibited by cyclosporin A, and the fraction of variant xcex1 remained unchanged or actually increased slightly during storage. The particles loaded with cyclosporin A proved more physically stable than drug-free lipid particles, as can be seen from the smaller particle size and lower growth of the particles during storage. Cyclosporin acts as a stabiliser of lipid particles, and produces greater physical stability of the lipid dispersion.
The use of a lipid as drug carrier matrix presents considerable advantages in toxicological terms too. Most active principles of lipid nanoparticles have GRAS status or are accepted as GRAS substances (GRAS=generally regarded as safe) [Food Additivesxe2x80x94GRAS substances, Food Drug Cosmetic Law Reports, Chicago, 1994]. All lipids in general, and all emulsifiers authorised for oral administration (e.g. tablets, capsules, pellets, oral solutions and suspensions) can be used for their preparation. Typical materials for the lipid matrix are glycerides of fatty acids present in foodstuffs and in the human body. The emulsifiers may be, for example, lecithin, sodium cholate, polysorbates such as polysorbate 80, or block copolymers such as Poloxamer 188 (an A-B-A block copolymer of polyethylene/polypropylene oxide with a relative mean molecular weight of 8350 g/mole, in which the mean relative molecular weight of the polyoxypropylene portion is 1750 g/mole and the polyoxyethylene portion is 80%). The said emulsifiers are even authorised for intravenous administration.
To sum up, in this invention a drug carrier or drug has been developed for an optimum treatment protocol with optimised blood levels, achieved with the use of ultrafine lipid particles loaded with cyclosporin A. Cyclosporin increases the physical quality of the particle dispersion by forming particularly fine particles with a high level of homogeneity. Physical stability also increases after the incorporation of cyclosporin into the lipid particles during storage of the particle dispersion.